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Thoracic Cancer ISSN 1759-7706

T E C H N O L O G I C A L N OTE

Cloning short DNA into plasmids by one-step PCR

Cheng-Cheng Tao1†, Ying Yang1†, Fang Li2†, Ling Qiao3, Yue Wu1, Xiao-Dong Sun1,
Yuan-Yuan Zhang1,4 & Chang-Long Li1
1 West China School of Basic Medical Sciences & Forensic Medicine, Sichuan University, Chengdu, China
2 Department of Medical Oncology, Sichuan Cancer Hospital and Institute, School of Medicine, University of Electronic Science and Technology of China,
Chengdu, China
3 School of Basic Medicine, Chengdu University of Traditional Chinese Medicine, Chengdu, China 4
School of Biological Sciences and Technology, Chengdu Medical College, Chengdu, China

Keywords Abstract
Homologous recombination; one-step PCR; plasmid
Background: Plasmid construction of small fragments of interest (such as inser-
construction; restriction enzyme-based cloning
method. tion of small fragment marker genes, expression of shRNA, siRNA, etc) is the
basis of many biomolecular experiments. Here, we describe a method to clone
Correspondence short DNA into vectors by polymerase chain reaction (PCR), named one-step
Yuan-Yuan Zhang and Chang-Long Li, West PCR cloning. Our method uses PCR to amplify the entire circular plasmid. The
China School of Basic Medical Sciences & PCR was performed by the primers containing the gene of short DNA with over-
Forensic Medicine, Sichuan University,
lapping sequences between 10–15 bp. The PCR products were then transformed
Chengdu 610041, P.R. China.
Tel: +86 28 8550 1278
into E. coli and cyclized by homologous recombination in vivo.
Fax: +86 28 8550 1237 Methods: The pEGFP-N1-HA plasmid was constructed by one-step PCR and
Email: sarahyyzhang@hotmail.com (Zhang); transformation. Cells were transfected with pEGFP-N1-HA and pEGFP-N1
changlongli@scu.edu.cn (Li) plasmid using TurboFect transfection reagent. Protein expression was detected by
western blotting and the HA-GFP fusion protein was detected by confocal
†
These authors contributed equally microscopy.
Results: The pEGFP-N1-HA plasmid was successfully constructed and HA
Received: 6 August 2020;
Accepted: 30 August 2020. doi:
expression in cells.
Conclusions: Free from the limitations of restriction enzyme sites and omitting
10.1111/1759-7714.13660 the ligation process, our method offers a flexible and economical option of plas-
mid construction.
Thoracic Cancer 11 (2020) 3409–3415

Key points
Significant findings of the study A method to clone short DNA into plasmids
was found.
What this study adds: Our study provides a flexible and economical option to
clone short DNA into plasmids.

Introduction
Plasmid vectors with small or multiple-tags, and small
RNAs are commonly used in biomedical studies. Current
methods of plasmid construction require obtaining the
gene of interest from the amplification of the DNA tem-
plate or chemically synthesized oligonucleotides.1–3
Obtaining a gene fragment of interest from the DNA
library is the most commonly used method. 1 It relies on
digestion of DNA by type II restriction enzymes, and cut-
ting the plasmid DNA circles with the same restriction
nuclease to create linear DNA molecules. Finally, the gene
of interest and the linearized vector are separately purified,
mixed together at a certain molar ratio, and ligated with
T4 DNA ligase to form recombinant DNA circles. 4–7 This
process involves cumbersome subcloning procedures
including designing primers, digestion, ligation and verifi-
cation of recombinant plasmids. 8, 9 Therefore, its efficiency
is dependent on both the restriction enzyme and the DNA
ligase. In addition, unwanted nucleotide sequences may be
added to the insert, and result in undesirable changes in
the final products.10 The chemical synthesis method is also
called oligo overlap cloning, which can be used to add a

Thoracic Cancer 11 (2020) 3409–3415 © 2020 The Authors. Thoracic Cancer published by China Lung Oncology Group and John Wiley & Sons Australia, Ltd. 3409 This is an open
access article under the terms of the Creative Commons Attribution-NonCommercial License, which permits use, distribution and reproduction in any medium, provided the original work is
properly cited and is not used for commercial purposes.
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Cloning short DNA into plasmids C.-C. Tao et al.

short stretch of DNA to a plasmid, such as adding short

arms, has been previously reported.10, 22 Employing the
tags (HA-tag or Flag) and cloning shRNAs. 3, 11 The DNA
characteristics of E. coli, we developed a method for con-
frag- ment of interest with restriction site is obtained
structing plasmids based on one-step PCR and homolo-
through chemical synthesis, and the linearized vector with
gous recombination. Previously reported methods of
sticky ends is obtained by digesting the vector with
constructing plasmid using homologous recombination of
restriction endonu- clease.11, 12 Both methods share similar
E. coli are all for genes longer than 150 bp.22
limitations.
Here, we describe an efficient and flexible method based
To overcome these limitations, several PCR-based clon-
on one-step PCR and homologous recombination for con-
ing protocols have been proposed to skip the use of DNA
structing a plasmid containing the fragment of interest
ligases and restriction endonucleases. Similar to our
below 150 bp. We constructed the pEGFP-N1-HA plasmid
method, these methods are based on homologous recombi-
by inserting the target fragment HA-tag into the vector
nation. The PCR products are flanked by 15 to 60 bp
pEGFP-N1 for proof-of-principle validation.
sequences, which exactly match the ends of the linear vec-
tor.13 These include in vitro Gateway recombination clon-
ing technology,14 seamless ligation cloning extract
Methods
(SLiCE)15 and RecET,16 etc. The SLiCE method from a
commonly used laboratory E. coli strain, JM109 and
Cell culture
DH5α, can efficiently function as an effective alternative to
commercially available seamless DNA cloning kits.15 It Human 293T cells and HeLa cells were purchased from
also includes in vivo recombination methods, such as American Tissue Culture Collection (ATCC) and
relying on RecA recombination enzymes, Red/ET maintained in our laboratory. Cells were maintained in
recombination systems,17, 18 and DH5α competent cells.19 Dulbecco’s modified Eagle’s medium (HyClone, Logan
These methods require preparing the target gene and City, Utah, USA) containing 10% fetal bovine serum
linearized vector, (Biological Industries, Cromwell, USA) and 1% penicillin-
respectively. The target gene can also be inserted by the streptomycin in a humidified incubator with 5% CO2
QuikChange site-directed mutagenesis method.20 Similar and 37◦C.
to our one-step PCR method, the target fragment can be
inserted at any amplifiable site of the vector. The difference
is that when we designed the primers, we divided the target Construction of the pEGFP-N1-HA plasmid
sequence into three parts, the target sequence at the 5 0 end
The pEGFP-N1 vector (4733 bp) used in this study was
of the forward primer, the target sequence at the 5 0 end of
pro- vided by TaKaRa. The sequence of HA-tag was 5 0-
the reverse primer, and a 15 bp homology arm with over-
TAC- CCATACGACGTCCCAGACTACGCT-30 (27 bp).
lapped region for target gene. In the QuikChange site-
The
directed mutagenesis method, the sequence of the target
linearized vector pEGFP-N1 carrying the HA-tag was
fragment was designed at the 5 0 end of the forward primer,
obtained by PCR. The PCR products were verified by gel
while the 50 end of the reverse primer contained only a
electrophoresis. The original template plasmids were
15 bp homologous arm. Compared with the QuikChange
digested by DpnI enzyme (purified PCR products).
site-directed mutagenesis method, our one-step PCR
Linearized vectors were transformed into E. coli and self-
method reduces both the cost for primer synthesis and the
recombinant plasmids based on homologous
formation of primer dimers.
recombination.
Although DNA inserts below 90 bp can be inserted using
Sequences of primers of HA-tag were as following: for-
the SLiCE method,21 it is still difficult in most plasmid con-
ward, 50- TAC GAC GTC CCA GAC TAC GCT AAG
struction methods to insert short DNA fragments less than
CTT CGA ATT CTG CAG TCG AC-3 0; reverse, 50-CTA
150 bp.22 DNA inserts below 150 bp can be directly synthe-
CCG GAC TCA GAT CTC GAG CTC ATG TAC CCA
sized, but it is cost prohibitive with limited productivity. Also,
the effects of synthesizing two short oligonucleotides below TAC GAC GTC CCA-30. As for PCR reactions, each 20 μL
sample in the PCR thermocycler (Gene-Explorer Touch,
150 bp still depend on the efficiency of annealing, endonucle- Zhengzhou, China) contained 1 μL DNA template (30 ng/
ase, and ligase, etc. The fidelity also significantly decreases
μL), 1 μL forward primer (10 nM), 1 μL reverse primer
when synthesizing oligonucleotides between 100 bp and
(10 nM), 7 μL double distilled H2O, and 10 μL I-5 2× high
150 bp. Therefore, with these traditional methods it is espe-
fidelity master mix (MCLAB, San Francisco, USA). A
cially inappropriate to construct plasmids with DNA fragments nega-
below 150 bp. In this study, we amplified a linear vector tive control replacing master mix with double distilled
including the short target gene below 150 bp by one-step PCR. H2O was employed. The PCR cycling profile for all reac-
A DNA repair system in E. coli, homologous recombina- tions was as follows: initial denaturation at 98◦C for
tion, which can cyclize linearized vectors with homology 2 minutes, denaturation of template at 98 ◦C for
10 seconds, annealing at 64◦C for 15 seconds, and
3410 Thoracic Cancer 11 (2020) 3409–3415 © 2020 The Authors. Thoracic Cancer published by China Lung Oncology Group and John Wiley & Sons Australia, Ltd.
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C.-C. Tao et al. Cloning short DNA into plasmids

extension at 72 ◦C for 75 seconds, amplification for

18 cycles with the final extension at 72◦C for 1 minute.
The PCR products (4774 bp, containing homologous
sequences) were verified by electrophoresis on 0.9% aga-
rose gel. The remaining 17 μL PCR products were mixed
with 0.5 μL DpnI enzyme (BioLabs, Ipswich, USA), cen-
trifuged, and incubated at 37◦C for 3 hours.
Transformation of the pEGFP-N1-HA
plasmid into E. coli
E. coli competent DH5α (Transgene Biotech, Beijing,
China) (50 μL) was thawed on ice and mixed with 5 μL
PCR products. The mixture was then incubated on ice for
30 minutes and transferred into a water bath at 42◦C for
45 seconds followed by incubation on ice for 2 minutes.
The mixture was added to 1 mL prewarmed Luria Broth
(LB) and gently shaken at 37◦C and 150 rpm for
70 minutes. Finally, cells were plated on fresh LB-agar
plates supplemented with 50 μg/mL Kanamycin (BBI,
Shanghai China), and incubated overnight at 37◦C. The
monoclonal colonies were picked up and sequenced. The
length of the pEGFP-N1-HA plasmid was 4.76 kb.
A colony of transformed E. coli cells was picked and
grown in 1 mL LB at 37◦C for 4 hours. Then, 200 μL were
taken for DNA sequencing. The remaining cells were
added into 250 mL LB and incubated overnight at 37 ◦C.
Plasmid DNA was extracted with the GoldHi Endofree
Plasmid Miniprep Kit (Cwbio, Beijing, China) according to
the manufacturer’s instructions.

Figure 1 The construction of pEGFP-N1-HA plasmid. (a) The schematic diagram of traditional plasmid construction method based on overlapping oli- gonucleotides
and restriction enzymes. (b) Schematic diagram of our method based on one-step PCR and homologous recombination ( ) Over- lapping sequences of the templates (
) Homogenous arms. First, primers were designed and synthesized. The PCR-linear pairs were then obtained. Finally, the PCR products were transformed into
chemically competent E. coli. (c) Electrophoresis on SDS-SAGE gel of the PCR products. The size of the pEGFP-N1-HA plasmid was about 4.7 kb. (d) Diagram of
the PCR-linear pairs cyclization. Both the forward and reverse primer have a homolo- gous region, which can be circularized in competent E. coli ( ) Overlapping
sequences of the templates ( ) Homogenous arms ( ) HA-tag ( ) GFP.

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Cloning short DNA into plasmids C.-C. Tao et al.

Western blotting
Cells were transfected using TurboFect Transfection
reagent (Thermo, Waltham American) according to the
manufacturer’s instructions. Cells were lysed in ice-cold
lysis buffer 48 hours later, and lysates were centrifuged at
12000 rpm for 20 minutes at 4◦C. After measuring the pro-
tein content using a bicin-choninic acid (BCA) assay, equal
amounts of protein (1 g/mL) were electrophoresed by SDS-
PAGE on 12% acrylamide gels and transferred onto a
PVDF membrane. The membranes were blocked by 5%
skim milk in Tris-buffered saline with 0.1% Tween (TBST)
for 1 hour, and then incubated with anti-HA antibody
(Santa Cruz Biotechnology, Dallas, USA) and anti-GFP
antibody (Santa Cruz Biotechnology, Dallas, USA) at room
temperature for 2 hours. After three washes with TBST,
membranes were incubated with a secondary polyclonal
antibody to rabbit IgG conjugated with HRP at room tem-
perature for 1 hour. Labeled proteins were detected using
an ECL substrate (Mei5bio, Beijing, China).
Fluorescence imaging
Cells were fixed with 4% paraformaldehyde (Sangon,
Shanghai, China) for 20 minutes, permeabilized with 0.5%
PBS-Triton X-100 (BBI, Shanghai, China) for 18 minutes,
and blocked with 4% bovine serum albumin (BioFroxx,
Einhausen, Germany) in PBS at room temperature for
90 minutes. The slides were then incubated with anti-HA
antibody (Santa Cruz, Dallas, USA) for 2 hours at room
temperature, and subsequently incubated with secondary
antibody at room temperature for 2 hours. Nuclei were

Figure 2 Validation of our method. (a & b) The expression of GFP and HA-tag proteins in 293T cells transfected with pEGFP-N1 or pEGFP-N1-HA was detected
by western blotting, and quantified using ImageJ ( ) α-GFP ( ) α-HA. The values were normalized by the intensities of the corresponding α-tubulin band. (c)
The expression of GFP and HA proteins in 293A cells transformed with pEGFP-N1 or pEGFP-N1-HA was detected
by confocal microscopy. (d) Schematic diagram of our one-step PCR method for inserting large fragments. The large fragment insertion is segmented into a plurality
of fragments with a length of less than 150 bp according to a one-step PCR method. PCR-linear pairs with large fragment insertion are obtained by multiple
PCR amplification, digested by DpnI to eliminate the parent DNA, and directly transformed into competent E. coli. * indi-
cates P < 0.05, ** indicates P < 0.01, and *** indicates P < 0.001.

3412 Thoracic Cancer 11 (2020) 3409–3415 © 2020 The Authors. Thoracic Cancer published by China Lung Oncology Group and John Wiley & Sons Australia, Ltd.

C.-C. Tao et al.
stained with Hoechst (Sigma, Saint Louis, USA). Images
were captured using a confocal laser scanning microscopy
(Zeiss, Oberkochen, Germany).
Statistical analysis
All the data were normalized to the control. Data are
shown as mean SD and analyzed with t-test using the
GraphPad Prism (GraphPad, USA). P < 0.05 was consid-
ered statistically significant.
Results
pEGFP-N1-HA plasmid successfully
constructed using our method
Compared with the traditional construction method in
Fig 1a, the schematic map of our method is illustrated in
Fig 1b. The linearized vector was obtained by one-step
PCR. After annealing, the fragment of HA-tag was directly
cloned into the pEGFP-N1 vector. The new constructed
plasmid, pEGFP-N1-HA, was subjected to agarose-gel
elec- trophoresis. As shown in Fig 1c, PCR products were
absent in the negative control, while the pEGFP-N1-HA
reaction system showed a 4.7 kb PCR product. The
sequencing result of the pEGFP-N1-HA plasmid
construction was identical to the GenBank sequence (see
Fig 1d), which con- firmed the electrophoresis results.
HA-tag was expressed in 293T cells
The HA-tag expression in 293T cells was detected by west-
ern blotting. As shown in Fig 2a, HA-tag was successfully
expressed in cells. The HA-GFP fusion protein was clearly
detected by confocal microscopy (Fig 2b).
Discussion
To efficiently and rapidly construct plasmids with inserts
below 150 bp has prominent practical applications, such as
small peptide fusion proteins, small interfering RNAs
(siRNAs) or short hairpin RNAs (shRNAs). Traditional
plasmid construction depends on restriction enzymes and
ligases which are time-consuming and labor-intensive.
There have been many developments in constructing
recombinant vectors in vitro using recombinases and the
endogenous recombinases of E. coli. Seamless ligation
clon- ing extract (SLiCE) method is a commonly used
method, which utilizes bacterial cell extracts to assemble
multiple DNA fragments into recombinant DNA molecules
in a sin- gle in vitro recombination reaction. 23 Compared
with our method, the advantage of SLiCE is that SLiCE can
simulta- neously assemble multiple DNA fragments
without using
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Cloning short DNA into plasmids
recombinase, restriction enzyme and DNA ligase.
However, SLiCE involves the extraction of bacterial
solution and the preparation of linearized vectors and target
fragments. The recombination efficiency of different
bacterial cell extracts is also different. There have been
studies using the recom-
bination system of E. coli competent DH5α. This method
required neither engineering bacteria, nor recombinase and
bacterial cell extracts. However, the linearized vector and
the target fragment have to be prepared, respectively.
Therefore, these have to be digested by DpnI enzyme and
transformed.19, 24 The biggest difference between this
method and our method is that our method only requires
one step of PCR to obtain the linearized vector with the
target fragment. In addition, our method is also more flexi-
ble in position selection than the existing one-step PCR
method for constructing shRNA plasmid. 20 Our method is
based on homologous recombination, which is different
from the method for constructing site-directed mutagene-
sis25 and generation of shRNA expression vectors20 in pre-
vious reports. Compared to these previously reported
methods, our method is more flexible in selecting insertion
sites. The DNA fragment of interest can be inserted at any
position other than the promoter. Compared with direct
synthesis of short oligonucleotides, our method is more
economic. We also compared the cloning efficiency and
accuracy of constructing the pEGFP-N1-HA plasmid by
our one-step PCR method and the In-Fusion cloning
method. Our one-step PCR method synthesized 89 bp
DNA, about 47% less than 131 bp of the In-Fusion clon-
ing. There were more procedures in the In-Fusion cloning,
such as DNA fragment purification from PCR enzymatic
reactions, in vitro recombination, etc. According to the
sequencing results, there was the same accuracy between
our one-step PCR method and the In-Fusion cloning
method (data not shown).
Although our method was specifically designed for plas-
mids with small inserts below 150 bp, it can also be applied
to construct plasmids with large fragments (Fig 2c). Small
fragments can be obtained by PCR one by one, and ligated
to form a large DNA fragment. Our method has several
limitations. The efficiency may not be constant because the
plasmid template used in PCR amplification has some
transformation potential.25 In order to eliminate the influ-
ence of the plasmid template, it is necessary to digest the
template by DpnI endonuclease. The DpnI digestion time
of the PCR products plays key roles in the success of our
method. DNA polymerase may also induce errors. The
error rate may be high when PCR is used to amplify geno-
mic sequences. The amplified genomic sequences are lon-
ger, and the DNA polymerase mismatch rate is higher.26,
27
The use of high-fidelity Taq enzyme, such as Pfu DNA
polymerase,28 can reduce the mismatch rate in PCR, which
will also increase the cost.
3413

Cloning short DNA into plasmids
Overall, our method combines the advantages of both
PCR-based strategies and homologous recombination
(Figure S1). Due to its simplicity and versatility, our
method makes it possible to rapidly and cost-effectively
construct plasmids with inserts below 150 bp, which will
greatly contribute to the functional study of small peptides
and RNA interference.
Acknowledgments
Funding was provided by the National Natural Science
Foundation of China (Grant No. 81770580) and the
Sichuan Science and Technology Program (Grant
No. 2019YJ0050).
Disclosure
The authors declare that the research was conducted in the
absence of any commercial or financial relationships that
could be construed as a potential conflict of interest.
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Supporting Information
Additional Supporting Informationmay be found in the online
version of this article at the publisher’s website:
Figure S1. Supplementary Figure.

Thoracic Cancer 11 (2020) 3409–3415 © 2020 The Authors. Thoracic Cancer published by China Lung Oncology Group and John Wiley & Sons Australia, Ltd. 3415